Methods for detecting gene dysregulations

ABSTRACT

Described herein are methods, compositions and kits directed to the detection of gene dysregulations such as those arising from gene fusions and/or chromosomal abnormalities, e.g., translocations, insertions, inversions and deletions. Samples containing dysregulated gene(s) of interest may show independent expression patterns for the 5′ and 3′ regions of the gene. The methods, compositions and kits are useful for detecting mutations that cause the differential expression of a 5′ portion of a target gene relative to the 3′ region of the target gene.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/786,266, filed May 24, 2010, which claims the benefit of U.S.Provisional Application 61/181,217 filed on May 26, 2009, both of whichare hereby incorporated by reference in their entirely.

TECHNICAL FIELD

The present technology relates generally to detection of genedysregulations such as those arising from gene fusions and chromosomalabnormalities, which may be associated with various diseases. In aparticular aspect, the present technology relates to the detection ofgene deregulations using multiplex quantitative RT-PCR.

BACKGROUND

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present invention.

Variations in chromosome structure involve changes in parts ofchromosomes rather than changes in the number of chromosomes or sets ofchromosomes in the genome. There are four common types of mutations:deletions and duplications (both of which involve a change in the amountof DNA on a chromosome), inversions (which involve a change in thearrangement of a chromosomal segment), and translocations (which involvea change in the location of a chromosomal segment). All four classes ofchromosomal structure mutations are initiated by one or more breaks inthe chromosome. If a break occurs within a gene, then a gene mutationhas been produced, the consequence of which depends on the function ofthe gene and the time of its expression. Wherever the break occurs, thebreakage process leaves broken ends, which may adhere to other brokenchromosome ends or the normal ends of other chromosomes.

Reciprocal and Robertsonian translocations are the most frequentlyoccurring types of translocations. Reciprocal translocations usuallyinvolve a two-way exchange between different chromosomes. Thechromosomes break apart and segments below the break points swappositions. If the event is balanced, no net gain or loss of geneticmaterial results and the individual is usually phenotypically unaffectedif no genes are disrupted.

Robertsonian translocations occur when two chromosomes fuse at thecenters and essentially combine into one. Most of the genetic materialremains from both chromosomes. As in balanced reciprocal translocations,the carrier maybe normal, but produce genetically unbalanced gametes.Most progeny originating from unbalanced gametes do not survive and amiscarriage occurs during early pregnancy. If the carrier is fertile andprogeny survive, various defects could occur. One Robertsoniantranslocation results in the fusion of chromosomes 14 and 21. Resultingprogeny may inherit three copies of chromosome 21 which causes Down'ssyndrome.

Genetic abnormalities such as duplication, deletion, chromosomaltranslocation, and point mutation often lead to pathological conditions.Some diseases, such as cancer, are due to genetic abnormalities acquiredin a few cells during life, while in other diseases the geneticabnormality is present in ail cells of the body and present sinceconception.

SUMMARY OF THE INVENTION

Described herein are methods, compositions, and kits directed to thedetection of gene dysregulations such as those arising from gene fusionsand chromosomal abnormalities, translocations, insertions, inversionsand deletions. The methods, compositions and kits are useful fordetecting mutations that cause the differential expression of a 5′region of a target gene relative to the 3′ region of the target gene.

In one aspect, the present disclosure provides a method for detecting adysregulation in a target gene. The method may include: (a) amplifying a5′ region of the target gene transcript, if present, in a biologicalsample with one or more 5′ target primer pairs which are complementaryto the 5′ region of the target gene; (b) amplifying a 3′ region of thetarget gene transcript, if present, in the biological sample with one ormore 3′ target primer pairs which are complementary to the 3′ region ofthe target gene; and (c) detecting the amounts of amplification productproduced by the one or more 5′ target primer pairs and the one or more3′ target primer pairs. The method may also provide that a difference inthe amounts of amplification products produced by steps (a) and (b)indicates that the target gene is dysregulated.

In another aspect, the present disclosure provides a method furdetecting the presence or absence of a dysregulation in a target gene ina sample. The method may include: (a) measuring the amount oftranscription of a 5′ region of the target gene and a 3′ region of thetarget gene in the test sample; and (b) comparing the relativeexpression of the 5′ region to the 3′ region of the target gene in thetest sample to the relative expression of the 5′ region to the 3′ regionof the target gene in a reference sample. The method may also providethat a difference in the relative expression in the lest sample comparedto the reference sample is indicative of the presence of a genedysregulation. In an embodiment, the relative amount of transcript canbe determined using real-time PCR and comparing the threshold cycle, orCt, value, for each amplicon. The Ct value can be normalized to areference sample.

In another aspect, the disclosure provides a method fur diagnosingcancer or a susceptibility to cancer in a subject. The method mayinclude: (a) amplifying a 5′ region of the target gene transcript, ifpresent, in a biological sample with one or more 5′ target primer pairswhich are complementary to the 5′ region of the target gene; (b)amplifying a 3′ region of the target gene transcript, if present, in thebiological sample with one or more 3′ target primer pairs which arecomplementary to the 3′ region of the target gene; and (c) detecting theamounts of amplification product produced by the one or more 5′ targetprimer pairs and the one or more 3′ target primer pairs. The method mayalso provide that a difference in the amounts of amplification productsproduced by steps (a) and (b) indicates that the subject has cancer oris susceptible to cancer resulting from a gene dysregulation.

In another aspect, the disclosure provides a method for diagnosingprostate cancer or a susceptibility to prostate cancer in a subject. Themethod may include: (a) amplifying a 5′ region of the target genetranscript, if present, in a biological sample with one or more 5′target primer pairs which are complementary to the 5′ region of thetarget gene; (b) amplifying a 3′ region of the target gene transcript,if present, in the biological sample with one or more 3′ target primerpairs which are complementary to the 3′ region of the target gene; and(c) detecting the amounts of amplification product produced by the oneor more 5′ target primer pairs and the one or more 3′ target primerpairs. The method may also provide that a difference in the amounts ofamplification products produced by steps (a) and (b) indicates that thetarget gene is dysregulated.

In another aspect, the disclosure provides a method for diagnosingnon-small cell lung carcinoma (NSCLC) or a susceptibility to NSCLC in asubject. The method may include: (a) amplifying a 5′ region of thetarget gene transcript, if present, in a biological sample with one ormore 5′ target primer pairs which are complementary to the 5′ region ofthe target gene; (b) amplifying a 3′ region of the target genetranscript, if present, in the biological sample with one or more 3′target primer pairs which are complementary to the 3′ region of thetarget gene; and (c) detecting the amounts of amplification productproduced by the one or more 5′ target primer pairs and the one or more 3′ target primer pairs, the method may also provide that a difference inthe amounts of amplification products produced by steps (a) and (b)indicates that the target gene is dysregulated. Suitable target genesinclude, for example, ALK, and EML4.

Optionally, the nucleic acid sample containing the target gene ofinterest may be subjected to another analysis to determine the nature ofthe gene dysregulation. Suitable analyses include, for example,comparative hybridization (e.g., comparative genomic hybridization).Comparative hybridization techniques such as comparative genomichybridization (CGH) is limited by the fact that this technique is onlyable to detect unbalanced rearrangements (rearrangements that lead togain or loss of genetic material). Comparative hybridization cannotadequately detect chromosomal abnormalities such as balancedtranslocations. Thus, any of the methods of the invention may be used incombination with a comparative hybridization technique. In particular,the primary abnormality in most leukemias, lymphomas, and solid tumorsis a balanced translocation. The combination of the inventive methodswith comparative hybridization (e.g., CGH) will be able to detect bothbalanced and unbalanced rearrangements and provide a more accuratediagnosis than if the comparative hybridization technique was usedalone. In the case of unbalanced rearrangements, the comparativehybridization technique maybe used as a confirmatory assay. As discussedherein, target gene dysregulations may arise from gene fusions andchromosomal abnormalities including, for example, translocations,deletions, inversions, and insertions.

Suitable target genes for use with any of the foregoing methods include,for example, Transmembrane Protease Serine 2 (TMPRSS2), ETS Related Gene(ERG), ETS translocation variant 1 (ETV1), Solute Carrier Family 45,Member 3 (SLC45A3), Human Endogenous Retrovirus K (HERV-K_22q11.3),Chromosome 15 Open Reading Frame 21 (C15ORF21), Heterogeneous NuclearRibonucleoproteins A2/B1 (HNRPA2B1), ETS Translocation Variant 4 (ETV4),ETS Translocation Variant 5 (ETV5), Anaplastic lyimphoma kinase (ALK),or Echinoderm microtubule associated protein like 4 (EML4), EUS, RANBP2,PAX, BUS, COLIA1 CLTC, KIF5B FKHR, PDGFB, FEV, DDIT3, ATF1, CREA, SP3,NR4A3, WT1, SYT, SSX1, SSX2, SSX4, BCR, ABL, BCL2, RARA, NPM, and ATIC.

Any cancer or other disorder associated with a gene dysregulation may bediagnosed using any of the foregoing methods. Disorders suitable fordiagnosis include, for example, pediatric soft tissue sarcomas that haveindeterminate histologies.

In one embodiment, the biological sample is contacted with the one ormore 5′ target primer pairs and the one or more 3′ target primer in amultiplex amplification reaction. In one embodiment, the detecting isaccomplished using a labeled oligonucleotide probe complementary to eachamplification product. For example, each oligonucleotide probe mayinclude a different detectable label, such as a donor fluorophore andquencher moiety. In another embodiment, at least one of the primers forthe 5′ region and/or at least one of the primers for the 3′ region isdetectably labeled, preferably with different detectable labels. Inillustrative embodiments, the amplifying is performed using quantitativeRT-PCR, e.g., real-time RT-PCR.

In some embodiments, the chromosomal abnormality is selected from thegroup consisting of: a translocation, a deletion, an inversion, and aninsertion. In one embodiment, the biological sample is a sample from asubject to be tested for a chromosomal abnormality.

In one embodiment, the methods further include amplifying a region of anendogenous control gene transcript present in the biological sample witha primer pair complementary to the endogenous control gene and detectingthe amplification of the region of the endogenous control gene. In someembodiments, the amount of amplified target gene transcripts (i.e., the5′ region and the 3′ region) may be normalized to the amount ofamplified endogenous control gene transcript. Suitable endogenouscontrol genes include, for example, ABL.

In embodiments of any of the aspects herein, the method furtherincludes: (a) measuring the amount of transcription of a 5′ region of asecond target gene and a 3′ region of the second target gene in the testsample: and (b) comparing the relative expression of the 5′ region tothe 3′ region of the second target gene in the test sample to therelative expression of the 5′ region to the 3′ region of the secondtarget gene in a reference sample. The method may also provide that adifference in the relative expression of both the target gene and thesecond target gene in the test sample compared to the reference sampleis indicative of the presence of a target gene: second target genetranslocation. Exemplary target gene and second target genetranslocations include TMPRSS2:ERG, TMPRSS2:ETV1, and EML4:ALK.

Suitable biological samples include, for example, whole blood, isolatedblood cells, plasma, serum, and urine.

In another aspect, the disclosure provides a kit for detecting a geneticabnormality in a sample. The kit may include: (a) at least oneoligonucleotide for determining the level of expression of at least onesequence from the 5′ region of a target gene; and (b) at least oneoligonucleotide for determining the level of expression of at least onesequence from the 3′ region of the target gene. In one embodiment, thetarget gene is TMPRSS2 or ALK. In some embodiments, the kits furtherinclude one or more reagents for performing real-time RT-PCR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the quantitative RT-PCR design fordetection of TMPRSS2 translocations. Primers, designated by arrows, andprobes, designated by lines with circles representing fluorophore andquencher, designed to the 5′ and 3′ regions of TMPRSS2 are shown. PanelA: schematic representation of TMPRSS2 and ErythroblastTransformation-Specific (ETS) transcripts found in normal prostate alongwith the relative transcript level (High/Low). Panel B: schematicrepresentation of TMPRSS2:ETS fusion transcripts found in prostatetumors along with the relative transcript level (High/Low).

FIG. 2 is a plot showing TMPRSS2 IDE scores of FFPE tumor tissue from 25prostate cancer patients determined by the equation shown above with Ctvalues obtained from real-time RT-PCR. Results are grouped by the knownTMPRSS2:ERG fusion status confirmed by fluorescent RT-PCR (7 TMP:ERGnegative, 18 TMP:ERG positive). Two specimens from the TMPRSS2:ERGNegative group fall below 60 (encircled), one of which (M289) issuspected to have a TMPRSS2:ETV fusion based on ETV expression data(data not shown).

FIG. 3 are bar graphs showing the raw Intragenic Differential Expression(IDE) values for TMPRSS2 (FIG. 3A) and ERG (FIG. 3B) in controlspecimens (TMPRSS2:ERG prostate cancer cell line). Orientation of 5′ and3′ regions are shown by the raw IDE score (absolute value not applied)for TMPRSS2 (FIG. 3A) and ERG (FIG. 3B). Positive values indicate higher5′ levels while negative values indicate higher 3′ levels. Normalprostate RNA does not contain TMPRSS2 or ERG fusions, VCaP cell RNA ispositive for the TMPRSS2:ERG fusion.

FIG. 4 is a bar graph showing the TMPRSS2 IDE scores in FFPE tissue.Columns indicate average IDE scores from 14 BPH specimens and 30 PCaspecimens. Y-error bars represent standard error.

FIG. 5 is a bar graph showing the ERG IDE scores in FFPE tissue. Columnsindicate average IDE scores from 14 BPH specimens and 30 PCa specimens.Y-error bars represent standard error.

FIG. 6 shows The EML4-ALK fusions detected by direct by RT-PCR andfragment analysis. Right facing arrows indicate forward primers, leftfacing arrow indicates a reverse FAM-labeled primer. Expected sizes foreach variant are indicated in the table.

FIG. 7 is a chart comparing detection method results for ALK expressionand ALK rearrangement. The bar graph represents ALK expression (lightcolumns) and ALK IDE (dark columns) from four control cell lines and 32lung cancer tissue specimens. The dotted horizontal line indicates theIDE cutoff level. Results from the EML4:ALK fragment analysis, ALK IHC,and ALK FISH are shown in the table below the graph. Cells with indicatenegative result, Cells with “+” indicate positive result. Cells with “Q”indicate insufficient quantity.

DETAILED DESCRIPTION

Described herein are methods, reagents and kits for detecting genedysregulations such as those arising as a result of chromosomal orgenetic abnormalities in a sample, where the dysregulation leads todifferential expression or quantities of particular portions of targetgenes. Chromosomal abnormalities include, for example, translocations,deletions and assertions. Large-scale mutations can affect chromosomaland genetic structure and include, for example, deletions of largechromosomal regions, leading to loss of the genes within those regions;and translocations, which are mutations whose effect is to juxtaposepreviously separate pieces of DNA, potentially bringing togetherseparate genes to form functionally distinct fusion genes (e.g.,TMPRSS2-ERG, TMPRSS2-ETV and EML4-ALK). For example, deletions mayresult in apposing previously distant genes producing a fusion protein.Another example includes chromosomal inversions, which reverse theorientation of a chromosomal segment. All of these chromosomalabnormalities can disrupt or after coding sequences or elements in thenon-coding region that affect the level of transcription of a particularcoding sequence.

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to“an oligonucleotide” includes a plurality of oligonucleotide molecules,a reference to label is a reference to one or more labels, a referenceto probe is a reference to one or more probes, and a reference to “anucleic acid” is a reference to one or more polynucleotides.

As used herein, unless indicated otherwise, when referring to anumerical value, the term “about” means plus or minus 10% of theenumerated value.

The terms “amplification” or “amplify” as used herein includes methodsfor copying a target nucleic acid, thereby increasing the number ofcopies of a selected nucleic acid sequence. Amplification may beexponential or linear. A target nucleic acid may be cither DNA or RNA.The sequences amplified in this manner form an “amplification product.”While the exemplary methods described hereinafter relate toamplification using the polymerase chain reaction (PCR), numerous othermethods are known in the art for amplification of nucleic acids (e.g.,isothermal methods, rolling circle methods, etc.). The skilled artisanwill understand that these other methods may be used either in place of,or together with, PCR methods. See, e.g., Saiki, “Amplification ofGenomic DNA” in PCR Protocols, Innis et al., Eds., Academic Press, SanDiego, Calif. 1990, pp. 13-20; Wharam et al., Nucleic Acids Res.,29(11):E54-E54, 2001; Hafner et al., Biotechniques, 30(4):852-56, 858,860, 2001; Zhong et al., Biotechniques, 30(4):852-6, 858, 860, 2001.

As used herein, the term “detecting” refers to observing a signal from adetectable label to indicate the presence of a target nucleic acid inthe sample. The term detecting dots not require the method to provide100% sensitivity and/or 100% specificity. As is well known,“sensitivity” is the probability that a test is positive, given that thesubject has a target nucleic acid sequence, while “specificity” is theprobability that a test is negative, given that the subject does nothave the target nucleic acid sequence. A sensitivity of at least 50% ispreferred, although sensitivities of at least 60%, at least 70%, atleast 80%, at least 90% and at least 99% are clearly more preferred. Aspecificity of at least 50% is preferred, although sensitivities of atleast 60%, at least 70%, at least 80%, at least 90% and at least 99% areclearly more preferred. Detecting also encompasses assays with falsepositives and false negatives. False negative rates may be 1%, 5%, 10%,15%, 20% or even higher. False positive rates may be 1%, 5%, 10%, 15%,20% or even higher.

The terms “complement”, “complementary” or “complementarity” as usedherein with reference to polynucleotides (i.e., a sequence ofnucleotides such as an oligonucleotide or a genomic nucleic acid)related by the base-pairing rules. The complement of a nucleic acidsequence as used herein refers to an oligonucleotide which, when alignedwith the nucleic acid sequence such that the 5′ end of one sequence ispaired with the 3′ end of the other, is in “antiparallel association”.For example, for the sequence 5′-A-G-T-3′ is complementary to thesequence 3′-T-C-A-5′. Certain bases not commonly found in naturalnucleic acids may be included in the nucleic acids of the presentinvention and include, for example, inosine and 7-deazaguanine.Complementarity need not be perfect; stable duplexes may containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.Complementarity may be “partial” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete,” “total,” or “full” complementarity between thenucleic acids.

The term “detectable label” as used herein refers to a molecule or acompound or a group of molecules or a group of compounds associated witha probe and is used to identify the probe hybridized to a genomicnucleic acid or reference nucleic acid. In some cases, the detectablelabel may be detected directly. In other cases, the detectable label maybe a part of a binding pair, which can then be subsequently detected.Signals from the detectable label may be detected by various means andwill depend on the nature of the detectable label. Examples of means todetect detectable label include but are not limited to spectroscopic,photochemical, biochemical, immunochemical, electromagnetic,radiochemical, or chemical means, such as fluorescence,chemifluoresence, or chemiluminescence, or any other appropriate means.

A “fragment” in the context of a gene fragment or a chromosome fragmentrefers to a sequence of nucleotide residues which are at least about 10nucleotides, at least about 20 nucleotides, at least about 25nucleotides, at least about 30 nucleotides, at least about 40nucleotides, at least about 50 nucleotides, at least about 100nucleotides, at least about 250 nucleotides, at least about 500nucleotides, at least about 1,000 nucleotides, at least about 2,000nucleotides, at least about 5,000 nucleotides, at least about 10,000nucleotides, at least about 20,000 nucleotides, at least about 50,000nucleotides, at least about 100,000 nucleotides, at least about 500,000nucleotides, at least about 1,000,000 nucleotides or more.

The term “genetic abnormality” or “chromosomal abnormality” as usedherein refers to a deviation of the nucleic acid sequence from awild-type or normal genetic sequence. A genetic abnormality may reflecta difference between the full genetic complement of an organism, or anyportion thereof, as compared to a normal full genetic complement of allchromosomes in that organism. For example, a genetic abnormality mayinclude a change in chromosomes or a portion thereof (e.g., deletions,duplications, amplifications); or a change in chromosomal structure(e.g., translocations, point mutations). Genetic abnormality may behereditary, i.e., passed from generation to generation ornon-hereditary. Genetic abnormalities may be present in some cells of anorganism or in all cells of that organism.

The term “endogenous control gene” as used herein refers to genes thatare generally always expressed and thought to be involved in routinecellular metabolism. Endogenous control genes are well known and includesuch genes as ABL, glyceraldehyde-3-phosphate dehydrogenase (G3PDH orGAPDH), albumin, actins, tubulins, cyclophilin, hypoxanthinephosphoribosyltransferase (HRPT), L32, 28S, and 18S rRNAs. Detection ofendogenous control genes in a diagnostic assay may serve as a positivecontrol for the assay.

The terms “identity” and “identical” refer to a degree of identitybetween sequences, there may be partial identity or complete identity. Apartially identical sequence is one that is less than 100% identical toanother sequence. Partially identical sequences may have an overallidentity of at least 70% or at least 75%, at least 80% or at least 85%,or at least 90% or at least 95%.

As used herein, the terms “isolated”, “purified” or “substantiallypurified” refer to molecules, such as nucleic acid, that are removedfrom their natural environment, isolated or separated, and are at least60% free, preferably 75% free, and most preferably 90% free from othercomponents with which they are naturally associated. An isolatedmolecule is therefore a substantially purified molecule.

The term “multiplex PCR” as used herein refers to an assay that providesfor simultaneous amplification and detection of two or more productswithin the same reaction vessel. Each product is primed using a distinctprimer pair. A multiplex reaction may further include specific probesfor each product that are detectably labeled with different detectablemolecules.

As used herein, the term “oligonucleotide” refers to a short polymercomposed of deoxyribonucleotides, ribonucleotides or any combinationthereof. Oligonucleotides are generally between about 10, 11, 12, 13,14, 15, 20, 25, or 30 to about 150 nucleotides (nt) in length, morepreferably about 10, 11, 12, 13, 14, 15, 20, 25, or 30 to about 70 nt,and most preferably between about 18 to about 26 nt in length.

As used herein, a “primer” is an oligonucleotide that is complementaryto a target nucleotide sequence and leads to addition of nucleotides tothe 3′ end of the primer in the presence of a DNA or RNA polymerase. The3′ nucleotide of the primer should generally be identical to the targetsequence at a corresponding nucleotide position for optimal extensionand/or amplification. The term “primer” includes all forms of primersthat may be synthesized including peptide nucleic acid primers, lockednucleic acid primers, phosphorothioate modified primers, labeledprimers, and the like. As used herein, a “forward primer” is a primerthat is complementary to the anti-sense strand of dsDNA. A “reverseprimer” is complementary to the sense-strand of dsDNA. As used herein, a“5′ target primer pair” is at least one forward primer and at least onereverse primer that amplifies the 5′ region of a target nucleotidesequence. As used herein, a “3′ target primer pair” is at least oneforward primer and at least one reverse primer that amplifies the 3′region of a target nucleotide sequence.

An oligonucleotide (e.g., a probe or a primer) that is specific for atarget nucleic acid will “hybridize” to the target nucleic acid undersuitable conditions. As used herein, “hybridization” or “hybridizing”refers to the process by which an oligonucleotide single strand annealswith a complementary strand through base pairing under definedhybridization conditions. It is a specific, i.e., non-random,interaction between two complementary polynucleotides. Hybridization andthe strength of hybridization (i.e., the strength of the associationbetween the nucleic acids) is influenced by such factors as the degreeof complementary between the nucleic acids, stringency of the conditionsinvolved, and the T_(m) of the formed hybrid.

“Specific hybridization” is an indication that two nucleic acidsequences share a high degree of complementarity. Specific hybridizationcomplexes form under permissive annealing conditions and remainhybridized after any subsequent washing steps. Permissive conditions forannealing of nucleic acid sequences are routinely determinable by one ofordinary skill in the art and may occur, for example, at 65° C. in thepresence of about 6×SSC. Stringency of hybridization may be expressed,in part, with reference to the temperature under which the wash stepsare carried out. Such temperatures are typically selected to be about 5°C. to 20° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Equations forcalculating T_(m) and conditions for nucleic acid hybridization areknown in the art.

As used herein, an oligonucleotide is “specific” for a nucleic acid ifthe oligonucleotide has at least 50% sequence identity with a portion ofthe nucleic acid when the oligonucleotide and the nucleic acid arealigned. An oligonucleotide that is specific for a nucleic acid is onethat, under the appropriate hybridization or washing conditions, iscapable of hybridizing to the target of interest and not substantiallyhybridizing to nucleic acids which are not of interest. Higher levels ofsequence identity are preferred and include at least 75%, at least 80%,at least 85%, at least 90%, at least 95% and more preferably at least98% sequence identity. Sequence identity can be determined using acommercially available computer program with a default setting thatemploys algorithms well known in the art BLAST). As used herein,sequences that have “high sequence identity” have identical nucleotidesat least at about 50% of aligned nucleotide positions, preferably atleast at about 60% of aligned nucleotide positions, and more preferablyat least at about 75% of aligned nucleotide positions.

The terms “target nucleic acid,” “target gene” and “target sequence” areused interchangeably herein and refer to nucleic acid sequence which isintended to be identified. Target nucleic acids may include 5′ or 3′regions of a target gene or any other sequence of interest. Targetnucleic acids may represent alternative sequences or alleles of aparticular gene. Target nucleic acids can be double stranded or singlestranded, or partially double stranded, or partially single stranded ora hairpin molecule. Target nucleic acids can be about 1-5 bases, about10 bases, about 20 bases, about 50 bases, about 100 bases, about 500bases, about 1,000 bases, about 2,000 bases, 2,500 bases, about 3,000bases, about 3,000 bases, about 4,000 bases, about 5,000 bases, about7,500 bases, about 10,000 buses, about 20,000 bases, about 30,000 bases,about 40,000 bases, about 50,000 bases, about 75,000 bases, about100,000 bases, about 1,000,000 bases or more.

The term “transcript,” when referring to a target nucleic acid, refersto any nucleic acid that is representative of the genomic nucleic acidof a cell including, for example. RNA in any form (e.g., mRNA, pre-mRNA,and snRNA) and synthetic representations of such as cDNA.

The term “test sample” as used herein refers to a sample, which containsnucleic acid or is suspected of containing nucleic acid. In someembodiments, the nucleic acids in the test sample are for use inaccordance with the methods disclosed herein. In some embodiments, atest sample is a biological sample.

The term “biological sample” as used herein refers to a sample, whichcontains target nucleic acids or be used as a source of target nucleicacids for the methods of the invention. A biological sample may includeclinical samples (i.e., obtained directly from a patient) or isolatednucleic acids and may be cellular or acellular fluids and/or tissue(e.g., biopsy) samples. In some embodiments, a sample is obtained from atissue or bodily fluid collected from a subject. Sample sources include,but are not limited to, sputum (processed or unprocessed), bronchialalveolar lavage (BAL), bronchial wash (BW), whole blood or isolatedblood cells of any type (e.g., lymphocytes), bodily fluids,cerebrospinal fluid (CSF), urine, plasma, serum, or tissue (e.g., biopsymaterial). The term “patient sample” as used herein refers to a sampleobtained from a human seeking diagnosis and/or treatment of a disease.In the case where the subject is a fetus, the patient sample can be fromthe subject (i.e., fetus), amniotic fluid, or maternal (e.g. themother's blood).

As used herein, the term “subject” refers to a mammal, such as a human,but can also be another animal such as a domestic animal (e.g., a dog,cat, or the like), a farm animal (e.g., a cow, a sheep, a pig, a horse,or the like) or a laboratory animal (e.g., a monkey, a rat, a mouse, arabbit, a guinea pig, or the like). The term “patient” refers to a“subject” who is, or is suspected to be, afflicted with disease relatedto a chromosomal abnormality.

General Overview of the Technology

Disclosed herein are methods for detecting the presence or absence oftarget gene dysregulations in subjects based, at least in part, onresults of the testing methods of the present technology on a sample.The test samples disclosed herein are represented by, but not limited inany way to, e.g., blood (or a fraction of blood such as plasma, serum,or particular cell fractions), lymph, mucus, tears, saliva, cysticfluid, urine, semen, stool, CSF, ascites fluid, whole blood, and biopsysamples of body tissue, fine needle aspirate (FNA), bronchalvcolarlavage (BAL). This disclosure is also drawn, inter alia, to methods ofdiagnosing or monitoring cancer. The cancer can be lung or prostatecancer bone and soft tissue sarcomas, various leukemias and lymphomas.

The technology generally provides for the detection, measuring, andcomparison of gene expression of different regions of a target genewithin a test sample. Accordingly, the various aspects relate to thecollection, preparation, separation, identification, characterization,and comparison of the abundance of messenger RNA in a test sample. Thetechnology further relates to detecting and/or monitoring a samplecontaining a messenger RNA for a 5′ region of a target gene and a 3′region of a target gene. As used herein, the phrases “detecting theamount” or “detecting the level” refer to the quantity of transcriptfrom any gene or part of a gene, such as the 5′ region of a gene, a 3′region of the target gene, a reference gene. The amount can be expressedas a concentration, as a number of copies, or as a Ct value, forexample. The threshold cycle, or Ct, value is the cycle at which signalintersects a threshold value when performing real-time nucleic acidamplification.

Specimens that do not contain a chromosomal abnormality within a targetgene will demonstrate the same expression pattern, between the 5′ regionand the 3′ region because they are linked in a unimolecular fashion.However, when the target gene is affected by some genetic or chromosomalabnormality, the 5′ and 3′ regions may show independent expressionpatterns for the 5′ and 3′ regions. In the ease of a translocation, the5′ and 3′ regions will show different expression patterns because thesetwo regions are now unlinked on the chromosome.

More specifically, a gene that undergoes certain rearrangements willexhibit differential expression of the 5′ region relative to the 3′region. This occurs in situations where the 5′ region of a gene remainsunder the control of the gene's regulatory elements, e.g., thoseelements contained in the 5′ untranslated region (UTR). The 3′ region ofthe gene is juxtaposed so as to be under the control of differentregulatory elements or none at all. For these types of mutations, the 5′region of the gene (e.g., at least one sequence that is specific to the5′ region such that it occurs upstream of the mutation break point ordeletion site) is expressed according to the target gene's ownregulatory elements, while the 3′ region (e.g., at least one sequencethat is specific to the 3 ′ region of the gene that occurs downstream ofthe mutation break point or deletion site) will not be expressed, in thecase where the 3′ region is deleted, or translocated to a position thatis not actively expressed, or expressed at a level consistent with theregulatory elements of a different gene.

Thus, the methods provide for detecting these mutations that result inthe differential expression of the 5′ region of a gene relative to the3′ region of the gene. One example of this situation occurs in manyprostate cancer patients, who have a translocation of the TMPRSS2 genesuch that the 5′ region of the TMPRSS2 gene remains under the control ofthe robust TMPRSS2 promoter, and the 3′ region of the TMPRSS2 gene istranslocated such that it is expressed by the less robust ERG or ETVpromoter.

As used herein, the phrases “difference of the level” and “difference inamounts” refer to differences in the quantity of transcript from the 5′region of a gene compared to the quantity of transcript from the 3′region of the target gene. In one embodiment, a transcript from the 5′region of a gene is present at an elevated amount or at a decreasedamount in a sample compared to the amount of transcript from the 3′region of the target gene. In wild-type or normal cells, the quantity oftranscript of the 5′ region of the target gene and the quantity oftranscript from the 3′ region of the target gene is expected to be atequal or near-equal quantities. By equal quantity, it is meant that themeasured amounts of transcript or detectable signal (which correlates tothe amount of transcript) for the 5′ region and the 3′ region do notexhibit a statistically significant difference from the same comparisonin control samples. Methods for comparing these values are known tothose of skill in the art and include, but are not limited to, aStudent's t-test and ANOVA analysis. The artisan recognizes that,because of technical differences inherent in the detection methodologiesused herein, the amount of detectable signal from the 5′-region may notnecessarily be equal to the amount of detectable signal from the3′-region even though no chromosomal abnormality is present (i.e., bothregions remain linked in a unimolecular manner and under the control ofthe same regulatory elements).

Distinct 3′-target gene expression levels expected to be found insamples containing target gene translocations and those withouttranslocations can be established by normalizing the expression levelsof 3′ target gene to 5′ target gene. An IDE Score can be calculatedaccording to the following formula:

IDE Score=2^(−(Ct) ^(3′-target gene) ^()−(Ct) ^(5′-target) ^(gene))

wherein the Ct (threshold cycle) values can be obtained by RT-PCR.

In other embodiments, the 3′- and 5′-target gene measurements may benormalized to an endogenous control gene when calculating an IDE score.Some useful formulae include, for example:

(3′ Target)/(Control)−(5′ Target)/(Control),

or

(3′ Target)/(5′ Target),

or

Ln((3′ Target)/(Control))−Ln((5′ Target)/(Control))

In other embodiments, the measured amount of the 3′- and 5′-transcriptsin the test sample may be normalized to the level of the sametranscripts from a control sample, rather than an endogenous gene.

In some embodiments, if the mean amount of transcript or detectablesignal for the 5′ region and the 3′ region are within about 1 standarddeviation, within about 0.5 standard deviations, within about 0.2standard deviations, within about 0.1 standard deviations, or withinabout 0.01 standard deviations, then there may be no significantdifference between the two amounts. In this example, one could concludethat the 5′ and 3′ regions are expressed in a unimolecular fashion andthere is no chromosomal abnormality in the target gene.

On the other hand, if the mean amount of transcript or detectable signalfor the 5′ region and the 3′ region exceed about 1 standard deviation,about 1.5 standard deviations, about 2.0 standard deviations, or about2.5 stand deviations, then there may be a significant difference betweenthe two amounts. In this example, one could conclude that the 5′ and 3′regions are expressed under the control of different promoters (or oneregion may not be expressed at all), such that there is a chromosomalabnormality in the target gene.

The measured amounts of transcript or detectable signal (whichcorrelates to the amount of transcript) may be expressed as a “relativeamount” or “ratio” of the expression of the 5′ region of the target generelative to the 3′ region of the target gene. Relative amounts may be asingle value or a range of values. For example, a range of values may beused to generate a standard curve relationship between the relativeamount of detectable signal formed versus some other quantity (e.g.,number of mRNA molecules). If the ratio of the expression of the 5′region of the target gene relative to the expression of the 3′ region ofthe target gene is statistically less than or greater than 1, then achromosomal abnormality is detected. Where the ratio is less than 1, the3′ region of the target gene has been translocated to a genomic regionthat is more transcriptionally active than the native target gene. Wherethe ratio is greater than 1, the 3′ region has either been deleted ortranslocated to a genomic region that is less transcriptionally activethan the native target gene.

In some embodiments, a sample obtained from a subject is assayed todetermine the relative expression levels of the 5′ and 3′ regions of aparticular gene or nucleic acid sequence of interest. Real-time RT-PCR(real-time reverse transcript-polymerase chain reaction) is a sensitivetechnique for mRNA detection and quantitation. Compared to the two othercommonly used techniques for quantifying mRNA levels, Northern blotanalysis and RNase protection assays, RT-PCR can be used to quantifymRNA levels from much smaller samples. In fact, this technique issensitive enough to enable quantitation of RNA from a single cell.

One of skill in the art would know how to design oligonucleotide primersand probes that are used to detect differential 5′ and 3′ expressionfrom any gene of interest, provided the sequence of the gene of interestis known. The size of the primer will depend on many factors, includingthe ultimate function or use of the oligonucleotide. An oligonucleotidethat functions as an extension primer or probe, for example, will besufficiently long to prime the synthesis of extension products in thepresence of a catalyst, e.g., DNA polymerase, and deoxynucleotidetriphosphates.

Alternatively, an insertion or transposition event can lead to thedifferential expression of the 5′ region and the 3′ region of a targetgene. The insertion of, for example, a promoter or other regulatoryelement, or the transposition of a transposable element into the middleof the coding sequence of a gene of interest can create a situationwhere the 5′ region of the target gene is expressed at a different levelthan the 3′ region of the target gene.

Any such mutation that results in the differential expression of a 5′region of a target gene and the 3′ region of the target gene isdetectable according to the methods, compositions and kits describedherein. One of skill in the art would know how to directed, for example,RT-PCR primers to a 5′ region of a gene of interest that occurs at ornear the start of transcription, thereby ensuring a productcorresponding to a 5′ region that occurs downstream of a potentialchromosomal abnormality. One of skill in the art need only refer to theknown sequence of the target gene and known base-pairing rules todetermine an effective RT-PCR primer or primer pair. Likewise, one ofskill in the art could design a primer or primer pair directed to a 3′region of the gene of interest. In particular examples, where a knownchromosomal abnormality occurs, one of skill in the art is further aidedby the knowledge of a known mutation site, thereby allowing the designof primers that are at or near the mutation site, e.g., a primer orprimer pair could be designed immediately 5′ of the mutation site andimmediately 3′ of the mutation site; or the primer or primer pairs couldbe designed, for example, within about 5 nucleotides (nt) of themutation site on either side, within about 10 nt of the mutation site oneither side, within about 20 nt of the mutation site on either side,within about 50 nt of the mutation site on either side, within about 100nt of the mutation site on either side, within about 250 nt of themutation site on either side or within about 500 nt of the mutation siteon either side.

Chromosomal Abnormality: Types and Associated Diseases

A chromosomal abnormality may reflect a difference between the fullgenetic complement or any portion thereof, of an organism, as comparedto a normal full genetic complement of all chromosomes in that organism.For example, a genetic abnormality may include a change in chromosomalcopy number (e.g., aneuploidy), or a portion thereof (e.g., deletions,duplications, amplifications); or a change in chromosomal structure(e.g., translocations, point mutations). A genetic abnormality may leadto pathological conditions. While some diseases, such as cancer, are dueto chromosomal abnormalities acquired in a few cells during life, theterm “genetic disease” most commonly refers to diseases present in allcells of the body and present since conception. Genetic abnormalitiesmay be hereditary or non-hereditary.

Genetic duplication is any duplication of a region of the genomicsequence. It may occur as an error in homologous recombination, aretrotransposition event, or duplication of an entire chromosome.Duplication of a gene has been associated with several diseases such assome cases of pagetic osteosarcoma is associated with duplication of MYCgene (Sarcoma, 1(3-4):131-134, 1997), some cases of breast cancer areassociated with duplication of HER-2/neu gene (Ann Oncol., 12(suppl1):S3-S8, 2001), some cases of bladder tumor are associated withduplication of c-erb-2 gene (Cancer Res., 55:2422-2430, 1995).

A deletion (also called gene deletion, deficiency, or deletion mutation)is a genetic aberration in which a part of a chromosome or a sequence ofDNA is missing. Deletion is the loss of genetic material. Any number ofnucleotides can be deleted, from a single base to an entire piece ofchromosome. Deletions can be caused by errors in chromosomal crossoverduring meiosis. Deletions are associated with an array of geneticdisorders, including some cases of male infertility and two thirds ofcases of Duchenne muscular dystrophy, a deletion of part of the shortarm of chromosome 5 results in a syndrome called Cri du chat, also knownas “cry of the cat” syndrome.

A chromosome “translocation” is the interchange of parts betweennonhomologous chromosomes. It is generally detected through cytogeneticsor a karyotyping of affected cells. There are two main types,reciprocal, in which all of the chromosomal material is retained andRobertsonian, in which some of the chromosomal material is lost.Further, translocations can be balanced (in an even exchange of materialwith no genetic information extra or missing) or unbalanced (where theexchange of chromosome material is unequal resulting in extra or missinggenes).

A reciprocal translocation between chromosomes 9 and 22 resulting in acytogenetically distinct acrocentric chromosome termed the Philadelphiachromosome. This translocation fuses the BCR gene locus of chromosome 22and the proto-oncogene ABL locus of chromosome 9 to form a her/abloncogenic protein (Tefferi et al. Mava Clin Proc. 80(3):390-402, 2005).Although the Philadelphia chromosome was first associated with CML, itis now known to be an indicator of prognosis in other blood disorderssuch as acute lymphoblastic leukemia (ALL).

Translocations have been linked with other diseases. For example, thefusion of the CBP gene of chromosome 16 to the MLL gene of chromosome 11through a translocation between chromosomes 11 and 16 has beenassociated with leukemia (Zhang et al., Genes Chromosomes Cancer,41(3):257-65, 2004). Similarly, a translocation between chromosomes 8and 21, resulting in a fusion of the AML1 and ETO genes is involved innearly 15% of acute myeloid leukemia (AML) cases (Zhang et al., Science,305:1286-9, 2004). Further, a number of chromosomal translocations havebeen identified in various forms of lymphoma. For example, atranslocation between chromosomes 8 and 14 involving the c-myc gene isreported to be present in approximately 80-85% of Burkittlymphoma/leukemia cases (Vega et al., Arch Pathol Lab Med,127:1148-1160, 2003). A further example is a translocation that resultsin the fusion of the EML4 gene and ALK gene. This EML4-ALK fusiontranslocation has been associated with NSCLC (Permer, et al., Neoplasia,10(3):298-302, 2008). Exemplary EML4:ALK fusions include the chromosome2p inversion (inv(2)(p21:p23)) which has been identified in 3-7% of allNSCLCs and the at least 11 identified variants of EML4:ALKtranslocations. In certain embodiments, IDE methods disclosed hereinallow for detection of translocations irrespective of the chromosomalbreakpoint.

In another example, a fusion of the androgen-regulated gene TMPRSS2 andmembers of the ETS family of transcription factors (e.g., ERG, ETV1, andETV4) have been identified in prostate cancers. Recurrent gene fusionsof the 5′ untranslated region of TMPRSS2 to ERG or ETV1 were found inprostate cancer tissues with outlier expression (Tomlins, S. et al.,Science, 310:644-648, 2005). These gene fusions occur in the majority ofprostate cancers identified by PSA screening and are the drivingmechanism for overexpression of the three members of the ETStranscription factor family, either ERG (21q22.3), ETV1 (7p21.2), orETV4 (17q21). It was found that 23 of 29 prostate cancer samplesharbored rearrangements in ERG or ETV1. Cell line experiments suggestedthat the androgen-responsive promoter elements of TMPRSS2 mediate theoverexpression of ETS family members in prostate cancer. Considering thehigh incidence of prostate cancer and the high frequency of this genefusion, the TMPRSS2-ETS gene fusion is the most common geneticaberration so far described in human malignancies. ERG is the mostcommon fusion partner of the ETS genes with TMPRSS2. This gene fusion isconsidered to be an early event in prostate cancer development. Fusionstatus in prostate cancer may determine clinical outcome. The methods,compositions and kits described herein, therefore, are useful for thedetection of TMPRSS2 translocations that are prevalent in prostatecancer patients, thereby allowing to the detection of prostate cancer oran individual who is at risk for developing prostate cancer.

Genetic abnormalities may also be point mutations insertions, ordeletions. A point mutation, or substitution, is a type of mutation thatcauses the replacement of single base nucleotide with anothernucleotide. Insertion and deletion includes insertions or deletions of asingle base pair. Mutations in the gene or chromosome often areassociated with diseases such as sickle cell anemia, cystic fibrosis,hemophilia, phenylketonuria, spina bifida, etc.

Target Nucleic Acids and Primers

The methods of the present invention relate to the detection ofchromosomal abnormalities in a target gene by amplifying a 5′ region ofthe target gene transcript, if present, in a biological sample with oneor more 5′ target primer pairs which are complementary to the 5′ regionof the target gene; and amplifying a 3′ region of the target genetranscript, if present, in the biological sample with one or more 3′target primer pairs which are complementary to the 3′ region of thetarget gene. Such regions can be amplified and isolated by PCR usingoligonucleotide primers designed based on genomic and/or cDNA sequencesthat encompass the regions. Any target gene that is potentially affectedby chromosomal abnormalities could be assayed according to the methodsdescribed herein.

The term “5′ region” refers to the portion of a polynucleotide locatedtowards the 5′ end of the polynucleotide relative to the 3′ region, andmay or may not include the 5′ most nucleotide(s) of the samepolynucleotide. In the context of translocations, the 5′-region refersto a region that is in the 5′ direction or upstream of a translocationbreakpoint. In the context of the present methods, the 5′ region may belocated near the 5′ end of the transcribed portion of the target gene.In some embodiments, the 5′ region encompasses all or a portion of the5′ untranslated region (UTR) of the target gene. In other embodiments,the 5′ region is located downstream of the start codon (if the targetgene is a protein-coding gene); for example, at least 10, at least 50,at least 100, at least 200, or at least 500 nucleotides downstream ofthe stop codon. The sue of the 5′ region to be amplified can varydepending on the detection method chosen. In some embodiments, theprimers may be selected to amplify at least 10, at least 20, at least30, at least 50, at least 100, at least 200, or at least 500 nucleotidesin the 5′ region.

The term “3′ region” refers to the portion of a polynucleotide locatedtowards the 3′ end of the polynucleotide relative to the 5′ region, andmay or may not include the 3′ most nucleotide(s) of the samepolynucleotide. In the context of translocations, the 3′-region refersto a region that is in the 3′ direction or downstream of a translocationbreakpoint. In the context of the present methods, the 3′ region may belocated near the 3′ end of the transcribed portion of the target gene.In some embodiments, the 3′ region encompasses all or a portion of the3′ UTR of the target gene. In other embodiments, the 3′ region islocated upstream of the stop codon (if the target gene is aprotein-coding gene); for example, at least 10, at least 50, at least100, at least 200, or at least 500 nucleotides upstream of the stopcodon. The size of the 3′ region to be amplified can vary depending onthe detection method chosen. In some embodiments, the primers may beselected to amplify at least 10, at least 20, at least 30, at least 50,at least 100, at least 200, or at least 500 nucleotides in the 3′region.

When assessing known genetic abnormalities, the terms “5′-region” and“3′-region” are somewhat relative in that each region is selected to beon a different side of the detect (e g., breakpoint) that results in thegenetic abnormality. These regions may be selected for convenience orother substantive reasons (i.e., simultaneous assessment of otherabnormalities such as mutations (SNPs), deletions, insertions, and thelike) and need not be at the 5′- and 3′-termini, respectively, of thetranscript. It is preferable that, when assessing target nucleic acidsfor unknown transcripts (i.e., a specific breakpoint has not beenpreviously identified), the distance between the 5′ region and the 3′region for a particular target gene should be maximized to the greatestextent possible to allow for the detection of a variety of chromosomalabnormalities that may occur between the two regions. This strategymaximizes the possibility that any breakpoint associated with a geneticabnormality occur between the two regions. In one embodiment, one orboth of the 5′- and 3′-regions assessed by the methods of this inventionare located in the untranslated regions (UTRs) of the transcripts.Guidelines for selecting primers for PCR amplification are well known inthe art. See. e.g., McPherson et al., PCR Basics: From Background toBench, Springer-Verlag, 2000. A variety of computer programs fordesigning primers are available, e.g., Oligo (National Biosciences. Inc.Plymouth Minn.), MacVector (Kodak/IBI), and the GCG suite of sequenceanalysis programs (Genetics Computer Group, Madison, Wis. 53711).

Sample Preparation

Specimens from which target nucleic acids can be detected and quantifiedwith the methods of the present invention may be obtained from subjectsaccording to methods known to those of skill in the art. Specimens maybe taken from body tissue and fluids such as blood (including wholeblood, serum, and plasma), urine, cerebrospinal fluid (CSF), synovialfluid, pleural fluid, pericardial fluid, intraocular fluid, tissuebiopsies or endotracheal aspirates, sputum, stool, swabs from, e.g.,skin, inguinal, nasal and/or throat. Methods of obtaining test samplesand reference samples are well known to those of skill in the art andinclude, but are not limited to, aspirations, tissue sections, drawingof blood or other fluids, surgical or needle biopsies, collection ofparaffin embedded tissue, collection of body fluids, collection ofstool, and the like. In one embodiment, the test sample may be obtainedfrom an individual who is suspected of having a disease or a geneticabnormality. In some embodiments, specimens are tissue samples (biopsysamples) from a subject having or suspected of having a disease or agenetic abnormality.

The nucleic acid (DNA and/or RNA) may be isolated from the sampleaccording to any methods well known to those of skill in the art. Ifnecessary, the sample may be collected or concentrated by centrifugationand the like. The cells of the sample may be subjected to lysis, such asby treatments with enzymes, heat surfactants, ultrasonication orcombinations thereof. The lysis treatment is performed in order toobtain a sufficient amount of RNA derived from the cells of interest, ifpresent in the sample, to detect using RT-PCR. Nucleic acid need not beextracted, but may be made available by suitable treatment of cells ortissue such as described in US Patent Publication No. 2008/131876.

In one embodiment, mRNA or cDNA generated from mRNA or total RNA may beused. Various methods of RNA extraction are suitable for isolating theRNA. Suitable methods include phenol and chloroform extraction. SeeManiatis et al., Molecular Cloning, A Laboratory Manual, 2d, Cold SpringHarbor Laboratory Press, page 16.54 (1989). In addition kits forisolating mRNA and synthesizing cDNA are commercially available e.g.,RNeasy Protect Mini kit, RNeasy Protect Cell Mini kit from Qiagen.

In one embodiment, a dual RNA/DNA isolation method is used employing atrizol based reagent for initial isolation of RNA and DNA from patientsamples. Upon contact with patient samples, the phenol and high saltreagents in the trizol effectively inactivate any disease agent orsecondary disease agent that may be present in the patient sample. Afterthe RNA and DNA are isolated from the patient samples, a silica basedcolumn may be used to further isolate the RNA and DNA. The use of silicabased columns allows for wash steps to be performed quickly andefficiently while minimizing the possibility of contamination. The washsteps may be used to remove PCR and RT-PCR inhibitors. The column methodfor nucleic acid purification is advantageous as it can be used withdifferent types of patient samples and the spin and wash stepseffectively remove PCR or RT-PCR inhibitors.

Amplification of Nucleic Acids

Nucleic acid samples or target nucleic acids may be amplified by variousmethods known to the skilled artisan. In suitable embodiments, PCR isused to amplify nucleic acids of interest. Briefly, in PCR, two primersequences are prepared that are complementary to regions on oppositecomplementary strands of the marker sequence. An excess ofdeoxynucleotide triphosphates are added to a reaction mixture along witha DNA polymerase, e.g., Taq polymerase.

In one embodiment, the target nucleic acids are amplified in a multiplexamplification reaction. A variety of multiplex amplification strategiesare known in the art and may be used with the methods of the invention.The multiplex amplification strategy may use PCR, RT-PCR or acombination thereof depending on the type of nucleic acid contained inthe disease agent(s). For example, if an RNA genome is present, RT-PCRmay be utilized. The PCR enzyme may be an enzyme with both a reversetranscription and polymerase function. Furthermore, the PCR enzyme maybe capable of “hot start” reactions as is known in the art.

If the target sequence is present in a sample, the primers will bind tothe sequence and the polymerase will cause the primers to be extendedalong the target sequence by adding on nucleotides. By raising andlowering the temperature of the reaction mixture, the extended primerswill dissociate from the target nucleic acid to form reaction products,excess primers will bind to the target nucleic acid and to the reactionproducts and the process is repeated, thereby generating amplificationproducts. Cycling parameters can be varied, depending on the length ofthe amplification products to be extended. An internal positiveamplification control (IC) can be included in the sample, utilizingoligonucleotide primers and/or probes.

Detection of Amplified Nucleic Acids

Amplification of nucleic acids can be detected by any of a number ofmethods well-known in the art such as gel electrophoresis, columnchromatography, hybridization with a probe, sequencing, melting curveanalysis, or “real-time” detection.

In one approach, sequences from two or more fragments of interest areamplified in the same reaction vessel (i.e., “multiplex PCR”). Detectioncan take place by measuring the end-point of the reaction or in “realtime.” For real-time detection, primers and/or probes may be detectablylabeled to allow differences in fluorescence when the primers becomeincorporated or when the probes are hybridized, for example, andamplified in an instrument capable of monitoring the change influorescence during the reaction. Real-time detection methods fornucleic acid amplification are well known and include, for example, theTaqMan® system, the Scorpion™ bi-functional molecule, and the use ofintercalating dyes for double stranded nucleic acid.

In end-point detection, the amplicon(s) could be detected by firstsize-separating the amplicons, then detecting the size-separatedamplicons. The separation of amplicons of different sizes can beaccomplished by, for example, gel electrophoresis, columnchromatography, or capillary electrophoresis. These and other separationmethods are well-known in the art. In one example, amplicons of about 10to about 150 base pairs whose sizes differ by 10 or more base pairs canbe separated, for example, on a 4% to 5% agarose gel (a 2% to 3% agarosegel for about 150 to about 300 base pair amplicons), or a 6% to 10%polyacrylamide gel. The separated nucleic acids can then be stained witha dye such as ethidium bromide and the size of the resulting stainedband or bands can be compared to a standard DNA ladder.

In another embodiment, two or more fragments of interest are amplifiedin separate reaction vessels. If the amplification is specific, that is,one primer pair amplifies for one fragment of interest but not theother, detection of amplification is sufficient to distinguish betweenthe two types—size separation would not be required.

In some embodiments, amplified nucleic acids are detected byhybridization with a specific probe. Probe oligonucleotides,complementary to a portion of the amplified target sequence may be usedto detect amplified fragments. Hybridization may be detected in realtime or in non-real time. Amplified nucleic acids for each of the targetsequences may be detected simultaneously (i.e., in the same reactionvessel) or individually (i.e., in separate reaction vessels). In someembodiments, the amplified DNA is detected simultaneously, using two ormore distinguishably-labeled, gene-specific oligonucleotide probes, onewhich hybridizes to the first target sequence and one which hybridizesto the second target sequence.

The probe may be detectably labeled by methods known in the art. Usefullabels include, e.g., fluorescent dyes (e.g., Cy5®, Cy3®, FITC,rhodamine, lanthamide phosphors, Texas red, FAM, JOE, Cal Fluor Red610®, Quasar 670®). ³²P, ³⁵S, ¹H, ¹⁴C, ¹²⁵I, ¹³¹I, electron-densereagents (e.g., gold), enzymes, e.g., as commonly used in an ELISA(e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkalinephosphatase), colorimetric labels (e.g., colloidal gold), magneticlabels (e.g., Dynabeads™), biotin, dioxigenin, or haptens and proteinsfor which antisera or monoclonal antibodies are available. Other labelsinclude ligands or oligonucleotides capable of forming a complex withthe corresponding receptor or oligonucleotide complement, respectively.The label can be directly incorporated into the nucleic acid to bedetected, or it can be attached to a probe (e.g., an oligonucleotide)that hybridizes or binds to the nucleic acid to be detected.

One general method for real time PCR uses fluorescent probes such as theTaqMan® probes, molecular beacons, and Scorpions™, Real-time PCRquantitates the initial amount of the template with more specificity,sensitivity and reproducibility, than other forms of quantitative PCR,which detect the amount of final amplified product. Real-time PCR doesnot detect the size of the amplicon. The probes employed in Scorpion™and TaqMan® technologies are based on the principle of fluorescencequenching and involve a donor fluorophore and a quenching moiety.

In one embodiment, the detectable label is a fluorophore. The term“fluorophore” as used herein refers to a molecule that absorbs light ata particular wavelength (excitation frequency) and subsequently emitslight of a longer wavelength (emission frequency). The term “donorfluorophore” as used herein means a fluorophore that, when in closeproximity to a quencher moiety, donates or transfers emission energy tothe quencher. As a result of donating energy to the quencher moiety, thedonor fluorophore will itself emit less light at a particular emissionfrequency that it would have in the absence of a closely positionedquencher moiety.

The term “quencher moiety” as used herein means a molecule that, inclose proximity to a donor fluorophore, takes up emission energygenerated by the donor and other dissipates the energy as heat or emitslight of a longer wavelength than the emission wavelength of the donor.In the latter case, the quencher is considered to be an acceptorfluorophore. The quenching moiety can act via proximal (i.e.,collisional) quenching or by Förster or fluorescence resonance energytransfer (“FRET”). Quenching by FRET is generally used in TaqMan® probeswhile proximal quenching is used in molecular beacon and Scorpion™ typeprobes.

In proximal quenching (a.k.a. “contact” or “collisional” quenching), thedonor is in close proximity to the quencher moiety such that energy ofthe donor is transferred to the quencher, which dissipates the energy asheat as opposed to a fluorescence emission. In FRET quenching, the donorfluorophore transfers its energy to a quencher which releases the energyas fluorescence at a higher wavelength. Proximal quenching requires veryclose positioning of the donor and quencher moiety, while FRETquenching, also distance related, occurs over a greater distance(generally 1-10 nm, the energy transfer depending on R-6, where R is thedistance between the donor and the acceptor). Thus, when FRET quenchingis involved, the quenching moiety is an acceptor fluorophore that has anexcitation frequency spectrum that overlaps with the donor emissionfrequency spectrum. When quenching by FRET is employed, the assay maydetect an increase in donor fluorophore fluorescence resulting fromincreased distance between the donor and the quencher (acceptorfluorophore) or a decrease in acceptor fluorophore emission resultingfrom decreased distance between the donor and the quencher (acceptorfluorophore).

Suitable fluorescent moieties include the following fluorophores knownin the art: 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid,acridine and derivatives (acridine, acridine isothiocyanate) AlexaFluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, AlexaFluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes),5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BlackHole Quencher (BHQ™) dyes (Biosearch Technologies), BODIPY® R-6G,BODIPY® 530/550, BODIPY® FL, Brilliant Yellow, coumarin and derivatives(coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151)), Cy2®, Cy3®, Cy3.5®,Cy5®, Cy5.5®, cyanosine, 4′,6-diaminidino-2-phenylindole (DAPI),5′,5″-dibromopyrogallol-sulfonephthalem (Bromopyrogallol Red),7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin,diethylenetriamine pentaacetate,4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid,4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid,5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL),4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), Eclipse(Epoch Biosciences Inc.), eosin and derivatives (eosin, eosinisothiocyanate), erythrosin and derivatives (erythrosin B, erythrosinisothiocyanate), ethidium, fluorescein and derivatives(5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), hexachloro-6-carboxyfluorescein(HEX), QFITC (XRITC), tetrachlorofluorescein (TET)), fluoreseamine,IR144, IR1446, Malachite Green isothiocyanate, 4-methylumbelliferone,ortho cresolphthalein, nitrotyrosine, pararosaniline, Phenol Red,B-phycoerythrin, R-phycoerythrin, o-phthaldialdehyde, Oregon Green®,propidium iodide, pyrene and derivatives (pyrene, pyrene butyrate,succinimidyl 1-pyrene butyrate), QSY® 7, QSY® 9, QSY® 21, QSY® 35(Molecular Probes), Reactive Red 4 (Cibacron® Brilliant Red 3B-A),rhodamine and derivatives (6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride,rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamineX isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonylchloride derivative of sulforhodamine 101(Texas Red)),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,tetramethyl rhodamine isothiocyanate (TRITC), CAL Fluor Red 610, Quasar670, riboflavin, rosolic acid, terbium chelate derivatives.

Other fluorescent nucleotide analogs can be used, see, e.g., Jameson,278 Meth. Enzymol., 363-390 (1997); Zhu, 22 Nucl. Acids Res., 3418-3422(1994). U.S. Pat. Nos. 5,652,099 and 6,268,132 also describe nucleosideanalogs for incorporation into nucleic acids, e.g., DNA and/or RNA, oroligonucleotides, via either enzymatic or chemical synthesis to producefluorescent oligonucleotides. U.S. Pat. No. 5,135,717 describesphthalocyanine and tetrabenztriazaporphyrin reagents for use asfluorescent labels.

The detectable label can be incorporated into, associated with orconjugated to a nucleic acid. Label can be attached by spacer arms ofvarious lengths to reduce potential steric hindrance or impact on otheruseful or desired properties. See, e.g., Mansfield, Mol. Cell. Probes.9:145-156 (1995). Detectable labels can be incorporated into nucleicacids by covalent or non-covalent means, e.g., by transcription, such asby random-primer labeling using Klenow polymerase, or nick translation,or amplification, or equivalent as is known in the art. For example, anucleotide base is conjugated to a detectable moiety, such as afluorescent dye, and then incorporated into nucleic acids during nucleicacid synthesis or amplification.

With Scorpion™ probes, sequence-specific priming and PCR productdetection is achieved using a single molecule. The Scorpion™ probemaintains a stem-loop configuration in the unhybridized state. Thefluorophore is attached to the 5′ end and is quenched by a moietycoupled to the 3′ end. The 3′ portion of the stem also contains sequencethat is complementary to the extension product of the primer. Thissequence is linked to the 5′ end of a specific primer via anon-amplifiable monomer. After extension of the Scorpion™ primer, thespecific probe sequence is able to bind to its complement within theextended amplicon thus opening up the hairpin loop. This prevents thefluorescence from being quenched and a signal is observed. A specifictarget is amplified by the reverse primer and the primer portion of theScorpion™, resulting in an extension product. A fluorescent signal isgenerated due to the separation of the fluorophore from the quencherresulting from the binding of the probe element of the Scorpion™ to theextension product.

TaqMan® probes (Heid et al., Genome Res, 6:986-994, 1996) use thefluorogenic 5′ exonuclease activity of Taq polymerase to measure theamount of target sequences in cDNA samples. TaqMan® probes areoligonucleotides that contain a donor fluorophore usually at or near the5′ base, and a quenching moiety typically at or near the 3′ base. Thequencher moiety may be a dye such as TAMRA or may be a non-fluorescentmolecule such as 4-(4-dimethylaminophenylazo) benzoic acid (DABCYL). SeeTyagi et al., Nature Biotechnology, 16:49-53 (1998). When irradiated,the excited fluorescent donor transfers energy to the nearby quenchingmoiety by FRET rather than fluorescing. Thus, the close proximity of thedonor and quencher prevents emission of donor fluorescence while theprobe is intact.

TaqMan® probes are designed to anneal to an internal region of a PCRproduct. When the polymerase (e.g., reverse transcriptase) replicates atemplate on which a TaqMan® probe is bound, its 5′ exonuclease activitycleaves the probe. This ends the activity of the quencher (no FRET) andthe donor fluorophore starts to emit fluorescence which increases ineach cycle proportional to the rate of probe cleavage. Accumulation ofPCR product is detected by monitoring the increase in fluorescence ofthe reporter dye (note that primers are not labeled). If the quencher isan acceptor fluorophore, then accumulation of PCR product can bedetected by monitoring the decrease in fluorescence of the acceptorfluorophore.

In a suitable embodiment, real-time PCR is performed using any suitableinstrument capable of detecting fluoroscence from one or morefluorescent labels. For example, real time detection on the instrument(e.g., a ABI Prism® 7900HT sequence detector) monitors fluorescence andcalculates the measure of reporter signal, or Rn value, during each PCRcycle. The threshold cycle, or Ct value, is the cycle at whichfluorescence intersects the threshold value. The threshold value isdetermined by the sequence detection system software or manually. The Ctvalue may be correlated to the amount of initial template nucleic acidin the reaction.

In some embodiments, melting curve analysis may be used to detect anamplification product. Melting curve analysis involves determining themelting temperature of nucleic acid amplicon by exposing the amplicon toa temperature gradient and observing a detectable signal from afluorophore. Melting curve analysis is based on the fact that a nucleicacid sequence melts at a characteristic temperature called the meltingtemperature (T_(m)), which is defined as the temperature at which halfof the DNA duplexes have separated into single strands. The meltingtemperature of a DNA depends primarily upon its nucleotide composition.Thus, DNA molecules rich in G and C nucleotides have a higher T_(m) thanthose having an abundance of A and T nucleotides.

Where a fluorescent dye is used to determine the melting temperature ofa nucleic acid in the method, the fluorescent dye may emit a signal thatcan be distinguished from a signal emitted by any other of the differentfluorescent dyes that are used to label the oligonucleotides. In someembodiments, the fluorescent dye for determining the melting temperatureof a nucleic acid may be excited by different wavelength energy than anyother of the different fluorescent dyes that are used to label theoligonucleotides. In some embodiments, the second fluorescent dye fordetermining the melting temperature of the detected nucleic acid is anintercalating agent. Suitable intercalating agents may include, but arenot limited to SYBR™ Green 1 dye, SYBR™ dyes, Pico Green, SYTO dyes,SYTOX dyes, ethidium bromide, ethidium homodimer-1, ethidiumhomodimer-2, ethidium derivatives, acridine, acridine orange, acridinederivatives, ethidium-acridine heterodimer, ethidium monoazide,propidium iodide, cyanine monomers, 7-aminoactinomycin D, YOYO-1,TOTO-1, YOYO-3, TOTO-3, POPO-1, BOBO-1, POPO-3, BOBO-3, LOLO-1, JOJO-1,cyanine dimers, YO-PRO-1, TO-PRO-1, YO-PRO-3, TO-PRO-3, TO-PRO-5,PO-PRO-1, BO-PRO-1, PO-PRO-3, BO-PRO-3, LO-PRO-1, JO-PRO-1, and mixturethereof. In suitable embodiments, the selected intercalating agent isSYBR™ GREEN 1 dye.

By detecting the temperature at which the fluorescence signal is lost,the melting temperature can be determined. In the disclosed methods,each of the amplified target nucleic acids may have different meltingtemperatures. For example, each of these amplified target nucleic acidsmay have a melting temperature that differs by at least about 1° C.,more preferably by at least about 2° C., or even more preferably by atleast about 4° C. from the melting temperature of any of the otheramplified target nucleic acids.

Methods of Diagnosis

In one aspect, the methods described herein provide for diagnosingprostate cancer or a susceptibility to cancer in a subject. The term“diagnose” or “diagnosis” as used herein refers to the act or process ofidentifying or determining a disease or condition in an organism or thecause of a disease or condition by the evaluation of the signs andsymptoms of the disease or disorder. Usually, a diagnosis of a diseaseor disorder is based on the evaluation of one or more factors and/orsymptoms that are indicative of the disease. That is, a diagnosis can bemade based on the presence, absence or amount of a factor which isindicative of presence or absence of the disease or condition. Eachfactor or symptom that is considered to be indicative for the diagnosisof a particular disease does not need be exclusively related to theparticular disease, i.e., there may be differential diagnoses that canbe inferred from a diagnostic factor or symptom. Likewise, there may beinstances where a factor or symptom that is indicative of a particulardisease is present in an individual that does not have the particulardisease. The methods include, but are not limited to, prostate and lungcancer and translocations, insertions, inversions and deletionsassociated with those cancers.

In one embodiment, the expression level of the 5′ region of the TMPRSS2gene is compared to the expression level of the 3′ region of the TMPRSS2gene in a sample firm a subject, wherein a difference in the expressionlevels of the 5′ region of the TMPRSS2 gene and the 3′ region of theTMPRSS2 gene is indicative of prostate cancer or a susceptibility toprostate cancer in the subject.

Methods of Prognosis

In one aspect, the methods described herein provide a prognosis forcancer or in a subject. The term “prognosis” as used herein refers to aprediction of the probable course and outcome of a clinical condition ordisease. A prognosis of a patient is usually made by evaluating factorsor symptoms of a disease that are indicative of a favorable orunfavorable course or outcome of the disease. The term prognosis doesnot refer to the ability to predict the course or outcome of a conditionwith 100% accuracy. Instead, the skilled artisan will understand thatthe term “prognosis” refers to an increased probability that a certaincourse or outcome will occur; that is, that a course or outcome is morelikely to occur in a patient exhibiting a given condition, when comparedto those individuals not exhibiting the condition. A prognosis may beexpressed as the amount of time a patient can be expected to survive.Alternatively, a prognosis may refer to the likelihood that the diseasegoes into remission or to the amount offline the disease can be expectedto remain in remission. Prognosis can be expressed in various ways; forexample prognosis can be expressed as a percent chance that a patientwall survive after one year, five years, ten years or the like.Alternatively prognosis may be expressed as the number of years, onaverage that a patient can expect to survive as a result of a conditionor disease. The prognosis of a patient may be considered as anexpression of relativism, with many factors affecting the ultimateoutcome. For example, for patients with certain conditions, prognosiscan be appropriately expressed as the likelihood that a condition may betreatable or curable, or the likelihood that a disease will go intoremission, whereas for patients with more severe conditions prognosismay be more appropriately expressed as likelihood of survival for aspecified period of time. The methods include, but are not limited to,prostate and lung cancer.

A prognosis is often determined by examining one or more prognosticfactors or indicators. These are markers, such as the presence of aparticular chromosomal translocation, the presence or amount of which ina patient (or a sample obtained from the patient) signal a probabilitythat a given course or outcome will occur. The skilled artisan willunderstand that associating a prognostic indicator with a predispositionto an adverse outcome may involve statistical analysis.

In one embodiment, the expression level of the 5′ region of the TMPRSS2gene is compared to the expression level of the 3′ region of the TMPRSS2gene in a sample from a subject, wherein a difference in the expressionlevels of the 5′ region of the TMPRSS2 gene and the 3′ region of theTMPRSS2 gene is indicative of stage, severity or outcome of prostatecancer in the subject. Nam et al., Br J. Cancer, 97:16390-1695, 2007,examined prostate cancer specimens from 165 patients who underwentsurgery for clinically localized prostate cancer between 1998 and 2006.They tested for the presence of TMPRSS2:ERG gene fusion product andconducted a survival analysis to determine the prognostic significanceof the presence of the TMPRSS2:ERG fusion gene on the risk of prostatecancer recurrence, adjusting for the established prognostic factors. Thesubgroup of patients with the fusion protein had a significantly higherrisk of recurrence (58.4% at 5 years) than did patients who lacked thefusion protein (8.1%, P<0.0001). Among prostate cancer patients treatedwith surgery, the expression of TMPRSS2:ERG fusion gene is a strongprognostic factor and is independent of grade, stage and PSA level. Assuch, the present methods are useful in providing a prognosis forrecurrence of prostate cancer.

Example 1

The examples below illustrate a standard protocol for performing RT-PCRand analyzing in real time. The TaqMan system of probe labeling is anexemplary method of real time detection of PCR amplicons. The followingexamples serve to illustrate the present invention and is in no wayintended to limit the scope of the invention.

To detect the presence of translocations or fusions involving TMPRSS2,commonly found fused with ETS transcripts in genetic samples fromprostate cancer patients, an approach was taken that identifies breakageof the 5′ and 3′ portions of TMPRSS2 and the subsequent translocation ofthe 3′ portion to a region under the control of regulatory elements thatare less robust than the regulatory elements normally associated withTMPRSS2, ETS (E-Twenty Six) is a family of transcription factors whichinclude, for example, ERG and ETS Translocation Variants (ETV), whichinclude, for example, ETV1, ETV4 and ETV5). TMPRSS2:ERG and TMPRSS2:ETVtranslocations generally result in expression of a fusion transcriptcontaining at minimum the 5′ untranslated region of TMPRSS2 fused tocoding regions of ERG and ETV. With this in mind, a real-time RT-PCRassay was designed to separately analyze expression levels of the 5′ and3′ regions of TMPRSS2 (FIG. 1). Samples that do not contain atranslocation involving TMPRSS2 demonstrate the same expression patternbetween the 5′ and 3′ region because they are linked and under thecontrol of the same regulatory elements (e.g., those contained in the 5′untranslated region) (FIG. 1, Panel A). Samples containing a TMPRSS2translocation, however, show independent expression patterns for the 5′and 3′ region, regardless of the translocation partner, because the tworegions are now unlinked and under the control of different regulatoryelements (FIG. 1, Panel B). In the case of ERG and ETV translocations, aportion of 3′ TMPRSS2, which is normally expressed at high levels in theprostate, has a much lower expression level than in non-translocatedsamples because it has been fused with the ERG or ETV coding region andregulatory sequences, which normally confers lower level of ERG or ETVexpression in the prostate relative to the TMPRSS2 expression. Oneadvantage of this detection system is that the translocation partner forTMPRSS2 need not be identified a priori and separately assessed.

Distinct 3′-TMPRSS2 expression levels expected to be found in samplescontaining TMPRSS2 translocations and those without translocations canbe established by normalizing the expression levels of 3′ TMPRSS2 to 5′TMPRSS2. In this study, a TMPRSS2 an IDE Score was calculated accordingto the following formula:

IDE Score=2^(−(Ct) ^(3′-TMPRSS2) ^()−(Ct) ^(TMPRSS2-5′UTR) ⁾

wherein the Ct values were obtained by RT-PCR

FIG. 2 shows a dot plot of 25 formalin-fixed and paraffin-embedded(FFPE) tissue samples grouped by TMPRSS2:ERG fusion status. For example,using an IDE cutoff of 80, at least 84% of the tumor specimens (21/25)were accurately identified as fusion negative or positive by the assaysof the present invention. Because such a large percentage of individualswith prostate cancer harbor TMPRSS2:ETS fusions, the described assaywould be a beneficial tool to diagnose the large majority of prostatecancer cases regardless of the TMPRSS2 fusion type involved.

Example 2 RNA Extraction

RNA from formalin-fixed and paraffin-embedded (FFPE) tissue wasextracted by column purification (HighPure miRNA isolation kit, Roche)followed by DNase I digestion (Invitrogen). Plasma RNA was extracted asfollows: Study 1: 1 mL plasma from each donor was extracted byNucliSENS® easyMAG® (Biomerieux) followed by DNase I digestion(Invitrogen). Plasma extraction was further optimized in Study 2 asfollows: 2 mL plasma from each donor was extracted by NucliSENS®easyMAG® (Biomerieux) followed by DNase I digestion in conjunction withRNA concentration utilizing RNeasy mini kit (Qiagen).

Real-time RT-PCR

TaqMan primer and probe sets were designed to independently amplify 5′and 3′ regions of each gene (TMPRSS2 model shown in FIG. 1). In separatereactions, 5′ and 3′ transcript regions and an endogenous control wereamplified by real-time RT-PCR (RNA Ultrasense, Invitrogen; ABI 7900Sequence Detector, Applied Biosystems).

Intragenic Differential Expression (IDE) Profile Calculations Study 1

TMPRSS2 was initially analyzed in FFPE tissue from 20 patients (9prostate cancer (“PCa”) and 11 benign prostate hyperplasia (“BPH”)) andplasma from 42 patients (32 PCa and 10 BPH). IDE was expressed as aratio of 3′:5′ transcript levels which were determined by real-timeRT-PCR. A normal 3′:5′ ratio (≧30) was established by comparingnonmalignant cells to tumor cells from FFPE tissue. This cutoff wassubsequently used to identify abnormal ratios in plasma specimens.

Study 2

Detection methods, quantification methods, and IDE calculations werefurther optimized in the second study. Eight-point standard curvesranging from 1 to 150 ng of PC-3 RNA (Ambion) were used to extrapolatetranscript quantities from Ct values. The absolute values of thedifferences in 5′ and 3′ levels from the same transcript were calculatedusing the transcript region quantities as determined by standard curveand normalized to endogenous control (ABL) (Calculation:IDE=(5′/ABL-3′/ABL)-b; where b represents gene-specific normalizationvalue). Normal ranges were determined by analyzing results from normalprostate RNA and known TMPRSS2:ERG fusion positive specimens

TMPRSS2 IDE In FFPE Tissue

The initial study of 20 FFPE tissue specimens and 42 plasma specimensfrom patients with prostate cancer or BPH utilized a simple 3′:5′ ratiocutoff to determined TMPRSS2 Intragenic Differential expression vs.mutual expression of the 2 regions analyzed (Table 1). With a 3′:5′ratio cutoff of <30, in FFPE tissue, TMPRSS2 IDE was observed in 100%(9/9) prostate cancer specimens and 9% (1/11) BPH specimens. In plasma,early studies yielded 20 samples with RNA passing QC standards. Of these20 samples, TMPRSS2 IDE was observed in 47% (7/15) PCa, 60% (9/15) PCasamples were positive for 5′, 3′, or both regions of TMPRSS2, and 20%(1/5) BPH specimens were positive for 1 region of TMPRSS2.

TABLE 1 Initial IDE determinations of TMPRSS2 in BPH and PCa specimensSpecimen 3′ or 5′ Detected 3′:5′ ≧30 3′:5′ <30 3′:5′ = UD FFPE TissueBPH 100% (11/11) 91% (10/11) 9% (1/11) 0% (0/11) PCa 100% (9/9) 0% (0/9)100% (9/9) 0% (0/9) Plasma BPH 20% (1/5) NA NA 100% (5/5) PCa 60% (9/15)6.7 (1/15) 47% (7/15) 47% (7/15) UD, undetected (3′ only or no 5′ or3′).

Optimization of the entire assay provided a better means forquantification of IDE and the initial studies were repeated andexpanded. With the improved assay TMPRSS2 IDE was first evaluated innormal prostate and a confirmed TMPRSS2:ERG positive prostate cancercell line (VCaP, ATCC) (FIG. 3). As expected, normal prostate TMPRSS2and ERG showed no very low IDE scores whereas VCaP cells showed theexpected pattern of high positive TMPRSS2 score (meaning 5′>3′) and ahighly negative ERG score (meaning 3′>5′). For ease of analysis, furtherIDE scores are expressed as absolute values.

RNA was purified from 52 FFPE tissue samples (32 PCa. 14 BPH, 6initially diagnosed as BPH but upon further review were determined to beatypical or have PIN) and analyzed for TMPRSS2:ERG fusion by directfusion detection (TMPRSS2 exon 1:ERG exon 4) and by TMPRSS2 IDE. TMPRSS2IDE scores were significantly higher in PCa (mean=1.6, SE=0.5) vs. BPH(mean=0.28, SE=0.06) (FIG. 4). Likewise, ERG IDE scores were alsosignificantly higher in PCa vs. BPH (FIG. 5). Direct TMPRSS2:ERG fusiondetection revealed 56% (18/32) positive PCa specimens and no positiveBPH (14) or atypical/PIN (6) specimens (Table 2). With a cutoff of 0.25(IDE>0.25), TMPRSS2 IDE was observed in 84% PCa (26/31), 67%Atypical/PIN (4/6) and 36% BPH (5/14) (Table 2).

TABLE 2 Detection of TMPRSS2 rearrangements by in FFPE tissueTMPRSS2:ERG TMPRSS2 IDE Diagnosis − + <0.25 >0.25 BPH 100% (14/14)  0%(0/14) 71% (10/14) 36% (5/14) Prostate  44% (14/32) 56% (18/32) 23%(7/31) 84% (26/31) Cancer Atypical/PIN 100% (6/6)  0% (0/6) 33% (2/6)67% (4/6)

TMPRSS2 Detection in Plasma

Plasma specimens were also assayed for the presence of TMPRSS2:ERG, and5′ UTR and 3′ coding regions of TMPRSS2. RNA was extracted from 1 ml(Study 1) or 2 mL (Study 2) plasma from a total of 67 specimens (42 PCaand 17 BPH). Results from samples that sufficiently amplified endogenouscontrol are shown in Table 3. Clearly, Study 2 resulted in a higher rateof detection of 5′ UTR or 3′ coding region of TMPRSS2 with 78% (7/9)positive PCa and 0% (0/3) BPH as compared to 44% PCa and 17% BPH inStudy 1. Most notably however, analyzing expression of both 5′ UTR and3′ coding regions of TMPRSS2 increases the number of positive specimensby approximately 10-15% compared to detection of 5′ UTR or 3′ codingregion alone and demonstrates significant improvement over detection ofTMPRSS2:ERG fusion where only 1 positive PCa specimen was found.Overall, TMPRSS2 was detected in plasma from 44-78% of PCa and 0-17% ofBPH specimens.

TABLE 3 Detection of TMPRSS2:ERG fusion and 5′ and 3′ TMPRSS2 in plasmaTMPRSS2 Region Detected BPH PCa Study 1 6 27 TMPRSS2:ERG fusion 0% (0/6) 4% (1/27) 5′ UTR 0% (0/6) 37% (10/27) 3′ coding 17% (1/6)  30% (8/27)5′ UTR and 3′ coding 0% (0/6) 19% (6/27) 5′ UTR or 3′ coding 17% (1/6) 44% (12/27) Study 2 3  9 TMPRSS2:ERG fusion 0% (0/3)  0% (0/9) 5′ UTR 0%(0/3) 67% (6/9) 3′ coding 0% (0/3) 67% (6/9) 5′ UTR and 3′ coding 0%(0/3) 56% (5/9) 5′ UTR or 3′ coding 0% (0/3) 78% (7/9)

ERG and ETV1 IDE in FFPE Tissue

The TMPRSS2 IDE strategy was extended to ETS transcription factors. Inparticular, ERG demonstrated significant differences in PCa specimens(mean=14.6 SE=5.5) as compared to BPH (mean=0.27, SE=0.03) (FIG. 5).With a cutoff of 0.4 (IDE>0.4), ERG IDE was observed in 97% PCa (29/30),0% Atypical/PIN (0/5) and 7% BPH (1/14) (Table 4). ETV1 IDE (IDEscore>0.08) was less frequent in PCa, where it was found in 30% (9/30)of specimens, but was also observed 14% BPH (2/14) and 20% Atypical/PIN(1/5). All prostate cancer specimens were positive for at least one ofthe markers tested, 80% were positive for at least two of the markers,and 24% were positive for all three (Table 5). No BPH specimens werepositive for more than one marker.

TABLE 4 Detection of ERG and ETV1 IDE in FFPE Tissue ERG IDE ETV1 IDEDiagnosis <0.40 >0.40 ≦0.08 >0.08 BPH  93% (13/14)  7% (1/14) 86%(12/14) 14% (2/14) Prostate  3% (1/30) 97% (29/10) 70% (23/30) 30%(9/30) Cancer Atypical/PIN 100% (5/5)  0% (0/5) 80% (4/5) 20% (1/5)

TABLE 5 Frequency of Single or Multiple IDE Scores in FFPE Tissue IDEPanel Diagnosis Negative ≧1 Positive ≧2 Positive 3 Positive BPH 50%(7/14)  50% (7/14)  7% (1/14)  0% (0/14) Prostate  0% (0/30) 100%(30/30) 80% (24/30) 27% (8/30) Cancer Atypical/PIN 20% (1/5)  67% (4/6)20% (1/5)  0% (0/5)

TABLE 6  Amplification Primers and Probes for IDE Analysis 5′TMPRSS2 UTR 5′ TMPRSS2 TAGGCGCGAGCTAAGCAGGA (SEQ ID  Forward NO: 1) 5′TMPRSS2 CCTGCCGCGCTCCAGGCGG (SEQ ID  Reverse NO: 2) 5′ TMPRSS2 ProbeAGGCGGAGGCGGAGGGCGAGGGGC (SEQ ID NO: 3) 3′ TMPRSS2 3′ TMPRSS2TGGTGCGAGGGAAGCAAT (SEQ ID NO: Forward 4) 3′ TMPRSS2CACCCAATGTGCAGGTGGA (SEQ ID NO: Reverse 5) 3′ TMPRSS2 ProbeAAAGGAACTTGCCCTGAGCACTCC (SEQ ID NO: 6) 5′ ERG 5′ ERG ForwardCATCCGCTCTAAACAACCTCA (SEQ ID NO: 7) 5′ ERG ReverseGGCCATAATGCGATCAAGTT (SEQ ID  NO: 8) 5′ ERG ProbeCTTTCTGGTCAGAGAGAAGCAA (SEQ ID NO: 9) 3′ ERG 3′ ERG ForwardCCAGGTGAATCGCTCAAGGAA (SEQ ID NO: 10) 3′ ERG ReverseGGGCTGCCCACCATCTTC (SEQ ID NO: 11) 3′ ERG ProbeTCTCCTGATGAATGCAGTGTGGCC (SEQ ID NO: 12)

CONCLUSION

Intragenic differential expression (IDE) of TMPRSS2 as well as ERG aresignificantly higher in prostate cancer specimens as compared to normalprostate and BPH. By establishing a cutoff for normal vs. abnormal IDEscores, we were able to detect TMPRSS2 differences in FFPE tissue from77% of prostate cancer samples while present in 29% of BPH samples. Evenhigher sensitivity and specificity was achieved with ERG where elevatedIDE scores were detected in 97% of prostate cancer samples and only 1(7%) BPH specimen. The high percentage of PCa specimens with elevatedERG IDE scores may be attributed to translocations with TMPRSS2 as wellas other yet to be identified 5′ fusion partners such as those recentlyfound to be involved in ETV1 and ETV5 gene fusions, including the 5′UTRs from SLC45A3, HERV-K_22q11.3, C15ORF21, and HNRPA2B1 (Helgeson etal. 2008 and Tomlins 2007).

IDEs in TMPRSS2 and ETV1 showed unexpected patterns in some samples.Invariably, ERG IDE was in the orientation of the 3′ transcript regionbeing present at higher levels than 5′ levels, as would be expected fromthe understanding that the consequence of TMPRSS2 translocation (andother 5′ translocation partners) is an increase in levels of thepartnered ETS transcription factor. Alternatively, TMPRSS2 and ETV1seemed to demonstrate more complexity in that not only were the expectedIDE orientations observed, but the reverse orientations were alsoobserved in many samples, meaning that the 5′ region of ETV1 and the 3′region of TMPRSS2 were at higher levels than their respectivecounterpart. This difference may underlie the observation thatTMPRSS2:ETV1 translocations are rarely found when assaying directly forthe fusion. Due to these variations, the IDE values are expressed as anabsolute value to account for differences in both orientations. Notably,all BPH and Atypical/PIN specimens that were positive for ETV1 IDEdemonstrated the reverse orientation while both orientations wereobserved in prostate cancer samples. Additionally, over one quarter ofthe prostate cancer samples demonstrated IDE in both ETV1 and ERG. Thismay be due to the presence of multiple focal points or multipleclonalities in a single specimen. It is apparent however, that ERG IDEis observed primarily in confirmed prostate cancer and was only observedin one BPH specimen.

These plasma sample studies (Study 1 and Study 2 described above)demonstrated that 5′ or 3′ TMPRSS2 could be detected in 44% (Study 1)and 78% (Study 2) of specimens from prostate cancer patients, when bothregions were assessed, as compared to 30-37% (Study 1) and 67% (Study 2)when assaying for only one region of TMPRSS2. By assaying for bothregions of TMPRSS2 and by improving extraction and detection methods, wewere able to increase the number of plasma specimens in which TMPRSS2was detected. TMPRSS2 was detected in only one BPH specimen from Study 1and no BPH specimens in Study 2. The 5′ UTR and 3′ coding region ofTMPRSS2 in normal and BPH urine specimens have been successfullyamplified for evaluation using this IDE methodology.

Example 3

A fusion gene with transforming activity, echinoderm microtubuleassociated protein like 4-anaplastic lymphoma kinase (EML4-ALK), isfound in approximately 5% of NSCLCs of lung cancer patients. Thepresence of the EML4-ALK fusion can be predictive of the response ofthese patients to certain therapies. We applied the IDE methodology totest the ability of using IDE to identify patients with potential ALKtranslocation, not limited to know variants or fusion partners. Patientlung cancer tissue samples were analyzed using the IDE methodology bydetermining the ALK IDE cutoff value and then comparing the calculatedcutoff to the ALK IDE values from lung cancer tissue samples. Thepositive results were further analyzed by direct detection of EML4-ALKfusions using RT-PCR. Finally, a subset of the NSCLC positive sampleswere screened by immunohistochemistry (IHC) and/or fluorescence in situhybridization (FISH).

ALK IDE Cutoff Determination

ALK IDE was analyzed in lung cancer tissue samples from 56 NSCLCpatients. ALK IDE scores are expressed as a ratio of 3′5′ transcriptlevels determined by real-time RT-PCR. The ALK IDE scores werecalculated by, first, determining 5′ and 3′ ALK transcript levels usingRT-PCR and, second, normalizing those ALK transcript levels using thetranscript levels of an endogenous control (ABL)(IDE=5′-ALK/ABL-3′-ALK/ABL). Using an EML4:ALK fusion-positive cell line(NCI-H2228), a positive control ALK IDE 3′:5′ ratio score of 0.7 wasestablished. This ALK IDE cutoff value of 7 was subsequently used toidentify abnormal ratios in tissue specimens, indicating the presence ofALK rearrangement. Further verification of the methodology confirmedthat the ALK IDE value was 0.0 in two EML4-ALK negative NSCLC cell lines(NCI-H838 and NCI-H1299).

TABLE 7  ALK Amplification Primers and Probes for IDE Analysis 3′ALK Primers 3′ ALK Forward CCCAACTTTGCCATCATTTT (SEQ ID  NO: 13) 3′ALK Reverse GCAAAGCGGTGTTGATTACA (SEQ ID  NO: 14) 3′ ALK ProbeFAM-TGAATACTGCACCCAGGACC-BHQ  (SEQ ID NO: 15) 5′ ALK Primers 5′ALK Forward TGGCTTTTGACAATATCTCCA (SEQ ID  NO: 16) 5′ ALK ReverseTGCAGGATCTTGTCCTCTCC (SEQ ID  NO: 17) 5′ ALK ProbeAGCCTGGACTGCTACCTCAC (SEQ ID  NO: 18)

ALK IDE In Lung Cancer Tissue

Using the ALK IDE 3′:5′ ratio cutoff of >0.7, in EML4-ALKfusion-positive cell lines, a diagnostically positive ALK IDE wasobserved in 11% (6/56) of lung cancer tissue specimens. We tested thesesix positive samples for direct detection of EML4-ALK fusions byfragment analysis using in-house designed RT-PCR primer sets (FIG. 6).Of the six, EML4-ALK fusions were observed in 83% (5/6) of the samples.

Immunohistochemistry (IHC) and/or Fluorescence In Situ Hybridization(FISH)

A subset of the NSCLC positive samples were screened by IHC and/or FISH(subset results are shown in FIG. 7). The five confirmed ALK IDEpositive samples and an additional five samples with slightly tomoderate elevated levels of ALK transcript (i.e., ALK IDE>0.1) werefurther analyzed by FISH. Eight of the ten samples showed ALKrearrangement and/or ALK gene amplification. Three samples interpretedas having ALK rearrangements by FISH were also positive using ALK IDE.Two other ALK IDE positive samples (one confirmed and one unconfirmed bydirect RT-PCR) were interpreted as rearrangement negative by FISH.

CONCLUSIONS

The application of IDE methodology to ALK is useful for identificationof ALK expression and chromosomal rearrangement. ALK IDE accuratelycategorized all FISH-confirmed rearrangements as positive and detectedrearrangements in at least one other confirmed case not identified byFISH. The IDE methodology functions as a universal molecular assay fordetermining ALK rearrangements in multiple tumor types. This informationcan be further used by physicians in deciding appropriate therapies forpatients.

OTHER EMBODIMENTS

Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement and variation of the inventionsembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications, improvements and variations areconsidered to be within the scope of this invention. The materials,methods, and examples provided here are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention.

The invention has been described broadly and genetically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

Other embodiments are set forth within the following claims.

1.-20. (canceled)
 21. A kit comprising: (a) one or more 5′ target primerpairs that are complementary to a 5′ region of a target gene transcriptand are suitable for amplifying a 5′ region of the target genetranscript; (b) one or more 3′ target primer pairs that arecomplementary to the 3′ region of the target gene transcript and aresuitable for amplifying a 3′ region of the target gene transcript;wherein the target gene is dysregulated in a subject having cancer. 22.The kit of claim 21, wherein the dysregulation is a chromosomalabnormality.
 23. The kit of claim 22, wherein the chromosomalabnormality is selected from the group consisting of: a translocation, adeletion, an inversion, and an insertion.
 24. The kit of claim 23,wherein the translocation is a balanced translocation.
 25. The kit ofclaim 21, further comprising one or more reagents for performing anucleic acid amplification reaction.
 26. The kit of claim 21, whereinthe target gene is selected from the group consisting of: TMPRSS2, ERG,ETV1, SLC45A3, HERV-K_22q11.3, C15ORF21, HNRPA2B1, ETV4, ETV5, ALK,EML4, EUS, RANBP2, PAX, BUS, COL1A1, CLTC, KIF5B FKHR, PDGFB, FEV,DDIT3, ATF1, CREA, SP3, NR4A3, WT1, SYT, SSX1, SSX2, SSX4, BCR, ABL,BCL2, RARA, NPM, and ATIC.
 27. The kit of claim 21, wherein the targetgene is selected from the group consisting of TMPRSS2, ERG, ETV1, ALK,and EML4.
 28. The kit of claim 21, wherein the cancer is a leukemia, alymphoma, or a solid tumor.
 29. The kit of claim 21, wherein the canceris prostate cancer or non-small cell lung carcinoma (NSCLC).
 30. The kitof claim 21, wherein the subject can be diagnosed as having cancer or asusceptibility to cancer by (a) amplifying the 5′ region of the targetgene transcript in a biological sample from the subject using the one ormore 5′ target primer pairs; (b) amplifying the 3′ region of the targetgene transcript in the biological sample from the subject using the oneor more 3′ target primer pairs; (c) detecting the amounts ofamplification product produced by the one or more 5′ target primer pairsand the one or more 3′ target primer pairs; (d) comparing the relativeexpression of the 5′ region to the 3′ region of the target gene in thebiological sample to the relative expression of the 5′ region to the 3′region of the target gene in a reference sample; and (e) diagnosing thesubject as having cancer or a susceptibility to cancer when thecomparison of step (d) indicates that the target gene is dysregulated.31. The kit of claim 30, further comprising a labeled oligonucleotideprobe complementary to each amplification product of (c).
 32. The kit ofclaim 31, wherein each oligonucleotide probe comprises a differentdetectable label.
 33. The kit of claim 30, wherein the biological sampleis selected from the group consisting of whole blood, isolated bloodcells, plasma, serum, and urine.
 34. The kit of claim 30, wherein theamplifying is performed by real time RT-PCR.
 35. The kit of claim 30,wherein the amounts of the amplification products produced by steps (a)and (b) are each normalized to an amount of an endogenous control genetranscript.
 36. The kit of claim 35, wherein the endogenous control geneis ABL.
 37. The kit of claim 21, wherein the target gene is TMPRSS2, andwherein: (i) the one or more 5′ target primer pairs comprises the primerpair having the sequence of SEQ ID NOS: 1 and 2; and/or (ii) the one ormore 3′ target primer pairs comprises the primer pair having thesequence of SEQ ID NOS: 5 and
 6. 38. The kit of claim 21, wherein thetarget gene is ERG, and wherein: (i) the one or more 5′ target primerpairs comprises the primer pair having the sequence of SEQ ID NOS: 7 and8; and/or (ii) the one or more 3′ target primer pairs comprises theprimer pair having the sequence of SEQ ID NOS: 10 and
 11. 39. The kit ofclaim 21, wherein the target gene is ALK, and wherein: (i) the one ormore 5′ target primer pairs comprises the primer pair having thesequence of SEQ ID NOS: 13 and 14; and/or (ii) the one or more 3′ targetprimer pairs comprises the primer pair having the sequence of SEQ IDNOS: 16 and
 17. 40. The kit of claim 31, wherein the oligonucleotideprobe comprises the sequence of SEQ ID NOS: 3, 6, 9, 12, 15, or 18.